1. Field of the Invention
This invention relates to Lyme disease, and more specifically relates to an assy for serum antibodies against Lyme disease antigen in which crossreactivity of the antigen to other antibodies in the serum is reduced.
2. Background of the Invention
Borrelia burgdorferi, a species within the genus Borrelia and family Treponemataceae, is a tick-borne spirochete which has been identified as the causative agent of Lyme disease.
Several approaches to diagnosis of Lyme disease have been investigated. Diagnosis by histological identification of the spirochete is very difficult because the spirochete to present in tissue or body fluid in small numbers, even in many advanced cases, and isolation is even more difficult. Diagnosis by assay for Borrelia antigen is disclosed U.S. Pat. No. 4,888,276 to Shelbourne. In the Shelbourne assay, a urine sample from a patient suspected of having Lyme disease is combined with an anti-Borrelia antibody, monoclonal or polyclonal, raised against the antigen and the antigen-antibody complex is detected with an enzyme labeled anti-antibody.
Most reports on diagnosis of the disease have relied on assay of serum samples for anti-Borrelia antibodies present in a patient's serum in response to infection. Several types of assays have been developed including enzyme linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA).
At the present state of the art, ELISA and IFA assays of sera from patients in the early stages of the disease, some of whom may be clinically asymptomatic, is generally recognized to be unsatisfactory because of weak antibody response. Also, false positives arising from crossreactivity wit other antibodies in the serum decease the specificity of the assays. For example, Russell et al. in the Journal of Infectious Diseases, 149, 465 (1984) assayed sera from healthy individuals, patients with Lyme disease, and patients with other infections by ELISA and IFA and found significant crossreactivity unless sera from other treponemal-infected patients were excluded.
Likewise, Grodzicki et al. in the Journal of Infectious Disease, 157, 790 (1988) describes a comparative study of the effectiveness of an indirect ELISA assay and an immunoblot assay for diagnosis of early Lyme disease, and concludes that less false positives result with the immunoblot method.
Attempts to improve assays for Lyme disease by enrichment of Borrelia antigen or adsorption of the crossreacting antibodies with proteins have been reported. Thus, Coleman et al., in the Journal of Infectious Diseases, 155, 756 (1987) shows that assay for Lyme disease by ELISA is improved by removal of the outer envelope fraction of the spirochete, electrophoresis and Western blotting of the residue to isolate a flagellin-enriched protein fraction and use of this material as the capture antigen. Fawcett et al. absorbs crossreacting antibodies with E. coli to reduce nonspecific binding and false positives.
Raout et al., in the Journal of Clinicial Microbiology, 27, 2152 (1989) discloses reduction in crossreactivity of sera positive for leptospirosis, syphilis or human immunodeficiency virus in the micro IFA test. The Raout et al. method includes adsorption of crossreacting antibodies with an ultrasonicate of Reiter treponemes at 37.degree. C. for 60 minutes. On the other hand, Magnarelli et al. in the American Journal of Epidemiology, 127, 818, (1988) report that, to date there has been limited success in efforts to increase the specificity of ELISA for Lyme disease by adsorption with Borrelia hermsii or Reiter treponemes.
Craft et al., in the Journal of Infectious Diseases, 149, 789 (1984) compares ELISA and IFA assays which include 90 minute preadsorption at 23.degree. C. Craft et al. concludes that ELISA is more sensitive and specific than IFA, and ELISA without preadsorption of crossreacting antibodies is the best diagnostic test of Lyme disease.
A commercial Lyme disease assay kit sold by Zeus Scientific Inc. Raritan, N.J., utilizes the IFA technique and is claimed to be useful for confirmation of Lyme disease in its later stages. A commercial ELISA kit sold under the trade name IMMUNOCLONE.RTM. by Access Medical Systems is claimed to be more accurate than the leading commercially available laboratory methods.
In a copending application of common assignee herewith, a method of assay for Lyme disease is disclosed in which Lyme disease capture antigen is denatured to improve assay spcificity.
Lyme disease was first identified in 1975, and today is well-recognized as a growing menace. In spite of extensive research, no satisfactory assay for the disease has yet been advanced, and diagnosis still relies heavily on clinical observations. There is thus a need for a rapid and reliable assay of high sensitivity and specificity. The present invention is directed to fulfulling this need.